The post synaptic density (PSD) at excitatory glutamatergic synapses is a complex molecular machine (molecular weight greater than one billion Daltons) which is known to be a key site of information processing and storage. In order to explore the detailed molecular organization of the PSD, we have developed a new method to freeze-substitute hippocampal cultures and then examine them by EM tomography in thin sections. Tomography reveals that the core of the PSD is a large array of vertically oriented filaments that contain PSD-95. We also identify two major type of transmembrane structures at PSDs matching AMPA and NMDA receptors, which are both contacted by the PSD-95 containing vertical filaments. The other (c-terminus) ends of the vertical filaments are linked by horizonatally oriented filaments. Recently, we have discovered that one class of these horizontal filaments is ordered to form hexagons cross-linking the vertical filaments concentrated under the NMDA receptors. These findings show how the PSD-95 matrix can stabilize glutamate receptors, and at the same time could be remodeled by the addition of new receptors and their binding partners at the edges of the PSD. The idea that the scaffold stabilizes the PSD is now being explored by using EM tomography to determine the effects of knocking down members of the PSD-95 family of proteins. After PSD-95 is knocked down, patches of PSD competely unravel, demonstrating the central role of the PSD-95 scaffold. We have developed an alternative preparation for high resolution EM tomography of isolated PSDs that is compatible with immunolabeling to identify components of the PSD. This advance depends on the discovery of a high resolution negative stain compatible with tomography. We have now shown that the important kinase, CaMKII, can be recognized and mapped in isolated PSDs, and their dynamic aspects investigated as outlined in NS003113-02 LN. A new project using mass spectroscometry is aimed at the quantification of PSD components from the highly purified preparation using a synthetic gene approach to produce recombinant artificial proteins. A covalent cross-linking strategy is also being applied to identify nearest neighbor molecules in the PSD.